A current major focus of the laboratory is investigation of the physiological functions of the neuropeptide Tuberoinfundibular Peptide of 39 residues (TIP39) and its receptor, the Parathyroid Hormone 2 (PTH2) receptor. These molecules were discovered in this laboratory several years ago. In previous years of this project we mapped the neuroanatomical distributions of TIP39 and the PTH2 receptor. TIP39 is synthesized by three discrete groups of neurons, two at the caudal border of the thalamus and one in the brainstem. TIP39 synthesizing neurons project to several brain areas involved in the regulation of emotional function. These areas contain a matching distribution of the PTH2 receptor, and neurons in these regions project to the areas containing TIP39 neurons. Thus the system is ideally positioned to coordinate and modulate functions relevant to mental disorders. The hypothalamic paraventricular nucleus and the surrounding area is one of the regions of high TIP39/PTH2 receptor density. This region contains cells with a major role in regulation of the release of several pituitary hormones. One of the pituitary hormones controlled by paraventricular neurons is ACTH, which in turn controls glucocorticoid secretion from the adrenal gland. Function of this hypothalamo-pituitary-adrenal (HPA) axis is central to stress responses, and is likely to be involved in mood and anxiety disorders. We found that infusion of TIP39 within the hypothalamic paraventricular nucleus caused an increase in the blood level of corticosterone (which is the mouse glucocorticoid). Our experiments showed that the mechanism for this effect is probably via TIP39 acting on PTH2 receptors that are present on excitatory, glutamatergic, nerve terminals adjacent to cells that synthesize and release ACTH secretagogues (mainly CRH). Based on our neuroanatomical colocalization data, and an inhibitory effect of paraventricular TIP39 infusion on blood prolactin levels, our data suggest that TIP39 is likely to have a modulatory effect on many hypothalamic pituitary hormone releasing factors. TIP39 and the PTH2 receptor are present in several brain areas that are part of pain processing pathways. Pathways that convey information related to painful stimuli to the brain can be divided into a sensory-discriminitive pathway that contains information related to localization and intensity, and a pathway more involved in affective dimensions of pain. Our previous neuroanatomical studies showed that TIP39 and the PTH2 receptor are present in most of the relay stations of the affective, medial, pain pathway. They are also present in areas that play a major role in modulation of pain responses by circuits within the brain (descending modulation). Many of these brain regions, amygdalar nuclei in particular, are also involved in other affective functions. Mood and pain strongly affect each other, and it seems likely that the interactions that underlie this effect take place in brain regions that are part of both mood and pain regulating circuitry. During this reporting period we performed a set of experiments to evaluate the potential contribution of TIP39 to the supraspinal regulation of pain. We found that the responses in several nociceptive assays were reduced when TIP39 signaling was blocked, either by genetic deletion of TIP39, null mutation of the PTH2 receptor, or acute administration of a PTH2 receptor antagonist we developed previously. (Since pain is a perception that animals cannot communicate, tests that evaluate measurable responses to unpleasant stimuli are used, and the phenomenon they measure is referred to as nociception.) The PTH2 receptor antagonist was much more effective when infused into brain ventricles than when delivered to the spinal cord, and in the mutant mice there was an increase in the response latency in the hotplate test, which is more affected by supraspinal signaling than the tail-flick test, which did not differ between wild-type and mutant mice. Both of these observations suggest that TIP39 effects are greater at a supraspinal than spinal level. In addition we observed significantly greater stress-induced analgesia, which is primarily developed at a supraspinal level, in the three types of mice with inhibited TIP39 signaling than corresponding wild-type or control mice. Endocannabinoids (endogenous substances that act on marijuana receptors) contribute to the regulation of many processes, including pain, and are involved in some forms of stress-induced analgesia. We examined the potential involvement of endocannabinoid pathways in the altered nociception that occurs with blockade of TIP39 signaling using drugs that act on cannabinoid receptors. The differences between TIP39 or PTH2 receptor knockout mice and wild-type mice, and between control and TIP39 antagonist treated mice, were eliminated by administration of a cannabinoid receptor antagonist, rimonabant. This suggests that increased endocannabinoid function may underlie the antinociceptive effects of blockade of TIP39 signaling. There was also a decrease in cannabinoid receptor mRNA and an increase in the level of an enzyme involved in endocannabinoid degradation (fatty acid amide hydrolase, FAAH) in the basolateral amygdala which, based on how other receptors and enzymes are regulated, is consistent with increased endocannabinoid synthesis. TIP39 and the PTH2 receptor are present in some, but not all, of the regions where cannabinoids act, and these changes in endocannabinoid signaling molecules were not present in regions from which TIP39 and the PTH2 receptor are absent. This brings up the possibility that manipulation of the TIP39/PTH2 receptor system could activate endogenous pain reducing effects of endocannabinoids without some of the problematic effects of drugs that act directly on all cannabinoid receptors.